To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity. Methods The full cDNA of COUP-TFII was clon...To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity. Methods The full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint. The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA. Results COUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter. Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity. Conclusion The human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells展开更多
Arabidopsis (Arabidopsis thaliana) WRKY33 is a key transcription factor in pathogen-induced defense signaling, but its function in abiotic stresses remains largely unclear. In this study, we report on the use of a rev...Arabidopsis (Arabidopsis thaliana) WRKY33 is a key transcription factor in pathogen-induced defense signaling, but its function in abiotic stresses remains largely unclear. In this study, we report on the use of a reverse-genetic approach, as well as a yeast (Saccharomyces cerevisiae) expression system, to determine the role of WRKY33 in drought. A T-DNA insertion deletion mutant of WRKY33 is more sensitive to dehydration. Through genome-wide screening the target genes of WRKY33 in yeast, we identified 23 candidate genes including a drought tolerance gene CesA8. Further results revealed that WRKY33 repressed CesA8 expression through binding to the W-box elements of CesA8 distal promoter region and probably interacting with the transcriptional activator of CesA8, MYB46. These findings revealed the primary molecular mechanism underlying the function of WRKY33 in response to展开更多
文摘To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity. Methods The full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint. The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA. Results COUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter. Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity. Conclusion The human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells
文摘Arabidopsis (Arabidopsis thaliana) WRKY33 is a key transcription factor in pathogen-induced defense signaling, but its function in abiotic stresses remains largely unclear. In this study, we report on the use of a reverse-genetic approach, as well as a yeast (Saccharomyces cerevisiae) expression system, to determine the role of WRKY33 in drought. A T-DNA insertion deletion mutant of WRKY33 is more sensitive to dehydration. Through genome-wide screening the target genes of WRKY33 in yeast, we identified 23 candidate genes including a drought tolerance gene CesA8. Further results revealed that WRKY33 repressed CesA8 expression through binding to the W-box elements of CesA8 distal promoter region and probably interacting with the transcriptional activator of CesA8, MYB46. These findings revealed the primary molecular mechanism underlying the function of WRKY33 in response to
